PHYTOCHEMICAL STUDIES OF ENDOLICHENIC FUNGI ISOLATED FROM HYPOTRACHYNA INFIRMA (KUROK.) HALE

Objective: The aim of this study is to investigate of phytopharmaceutical importance of endolichenic fungi isolated from Hypotrachyna infirma (Kurok.) Hale. Methods: The lichen species were collected from Sholaiyar hills, Coimbatore and identified as Hypotrachyna infirma (Kurok.)Hale. From this lichen, 29 endolichenic fungi were isolated and 13 endolichenic fungi were identified. From the identified endolichenic fungi, 26 extracts were prepared by successive solvent extraction methods using Ethyl acetate and chloroform. Results: The phytochemical study revealed the presence of important constituents like Alkaloids, Tannins, Carbohydrates, Phenols, Protein, Terpenoids, Steroids, Glycosides Flavonoids and Saponins. From the 13 endolichenic fungi, only 5 endolichenic fungi (Nigrospora oryzae (Berkand Broome)Petch, Geotrichum candidum Link, Scytalidium lignicola pesante, Aspergillus oryzae(Ahlb.) cohn, Aspergillus niger Gr.) have more constitutents. These 5 endolichenic fungi have good results in Quantitative analysis also. Conclusion: Compared to ethyl acetate extracts Chloroform extracts showed very less concentration of the phytochemicals. From this study we conclutated Nigrospora oryzae (Berk and Broome) Petch gave the best results in both qualitative and quantitative compared to other endolichenic fungi. fungus, Ethyl acetate, Chloroform and phytochemical S test the presence of Tannins, Flavonoids, Steroids, Phenol, Terpenoids, Saponins, Carbohydrates, Glycosides, Protein.


INTRODUCTION
Lichen is a stable self-supporting association of a mycobiont (fungus) and a photobiont (alga, also houses fungi as endophytes and these are generally termed as endolichenic fungi. The term 'endolichenic fungi' was coined by for the fungi that live inside lichen thalli without producing any disease symptoms [1]. Lichens are known to synthesize a variety of secondary metabolites having varied activity in response to external environmental conditions. The use of lichens in medicine is due to their secondary metabolites that are unique compared to those of higher plants. In the history of endophytic research, the year 1977 is an important landmark year, because of the discovery of endophytic fungus Epichloe coenophiala (Neotyphodium coenophialum) from Festuca arundinacea which is the cause of "fescue toxicosis" [2].
The endolichenic fungal species investigated till date for the isolation of bioactive secondary metabolites belong to several geographical locations. The estimated global lichen diversity is about 20,000 [15] from this only a small number of lichen species have been screened for harvesting the endolichenic fungi with the potential to offer bioactive metabolites. Therefore one can assume the magnitude of prospective lichen diversity which is waiting to be unveiled. The test for the analysis of phytochemical compounds bring up the way to determine therapeutic drugs progressively [16].
Many researches were undergone based on endophytic fungi but less investigation was made on endolichenic fungi and their bioactive compounds. The aim of this work is to identify and analyse the phytochemistry of endolichenic fungi with two different solvent extract.

Collection of lichen
Lichen sample was collected from Sholaiyar, Valparai Hills, Coimbatore District, Tamilnadu. The lichen sample was identified by Dr. Sanjeeva Nayaka, Principal Scientist, CSIR-National Botanical Research Institute, Lucknow, India. The collected lichen material was identified as Hypotrachyna infirma (Kurok.) Hale. This lichen was deposited at CSIR-National Botanial Research Institute, Lucknow, India with the Voucher number 36008.

Surface sterilization of lichen
Different types of protocols are available for surface sterilization. For this study modified protocol of [17] was used.
Healthy-looking macro lichen thallus was washed in tap water to remove all debris. Then the lichen was subjected to repeated washing in double distilled water to remove bryophytes/mosses and all other visible contamination. After this the washed samples were subjected to chemical surface sterilization by dipping them in 30% Hydrogen peroxide (H2O2 After the chemical surface sterilization, the samples were rinsed in sterile double distilled water twice and dried under aseptic conditions and were cut into small segments (0.5 cm × 0.5 cm).

Isolation of endolichenic fungi
The sterilized lichen samples were placed on petriplates containing Potato Dextrose Agar (PDA) media and sealed using parafilm.
These petriplates were incubated in the culture room at 25±1 °C until fungal growth was initiated. After initiation, the growing fungal mycelia tips were transferred to new PDA plates for obtaining pure culture. After 15 d, those endophytes that had grown on the PDA media were identified based on the morphology at National Fungal Culture Collection of India (NFCCI)-A National Facility, Pune, India.

Fungal extraction
A fraction of fungal isolates was transferred into Potato Dextrose Broth (PDB) by aseptically scraping using an inoculation loop. The isolates were transferred into conical flasks containing the Potato Dextrose Broth. These conical flasks were incubated in room temperature over a period of time. After 28 d, culture liquid was filtered with help of Whatman No. 1 filter paper. The filtrates were used for further phytochemical studies.
To the filtrate equal volume of solvents were added, mixed well for 10 min and kept for 5 min. Till the two clear immiscible layers were formed. The upper layer of the solvent containing the extracted compounds was separated using separating funnel. Solvent was evaporated and the compound was dried using a rotary vacuum evaporator to yield the crude metabolite [18]. The crude extract was then dissolved in Dimethyl sulphoxide at 1 mg/ml of concentration and the extract was kept at 4 °C.

Qualitative chemical evaluation
The different fungal filtrates thus obtained were qualitatively tested for the presence of the following phytochemical constituents. The ethyl acetate and chloroform extracts of 13 endolichenic fungi were subjected to preliminary phytochemical screening. All preliminary phytochemical study was carried using the methodologies of Auwal  [19][20][21][22][23] To test the presence of Tannins, Flavonoids, Steroids, Phenol, Terpenoids, Saponins, Carbohydrates, Glycosides, Protein.

Quantitative chemical evaluation
Based on the qualitative analysis, the quantitative study was carried out for Tannins, Flavonoids, Phenols, and saponins.

Total tannin content
Total tannin content was measured by Folin-Denis method [24] 50 µl of the extract was made up to 7.5 ml by adding double distilled water. Then 0.5 ml of Folin-Denis reagent and 1 ml of sodium carbonate were mixed with it. Again, volume was made up to 10 ml by adding double-distilled water. Absorption was recorded at 700 nm as the vanillin reagent will react with any phenols that have an un-substituted resorcinol or phlorglucinol nucleus and forms a coloured substitution product which is measured at 700 nm.

Total phenol content
Total phenolics were quantified and expressed as gallic acid equivalents according to the method proposed by Singleton et al. [25]. About 3.9 ml of distilled water and 0.5 ml of Folin-ciocalteau reagent were added to 0.1 ml of sample extract in a tube and incubated at room temperature for 3 min after which 2 ml of 20% sodium carbonate was added to this and kept at boiling water bath for 1 minute. Phenols react with phosphomolybdic acid in the Folinciocalteau reagent in alkaline medium and produce a blue coloured complex (molybdenum blue) that can be estimated calorimetrically at 650 nm.

Total flavonoid content
Total flavonoid content was measured by the aluminium chloride colorimetric method [26]. 1 ml of extract and standard solution of catechin (100 mg/ml) was added to 10 ml volumetric flask containing 4 ml of distilled water. To this 0.3 ml of 5% sodium nitrate was added. After 5 min, 0.3 ml of 10% aluminium chloride was added. Then after 1 minute, 2 ml of 1M sodium hydroxide was added and the total volume was made up to 10 ml with distilled water. The solution was mixed and the absorbance was measured against prepared reagent blank at 510 nm.

Total saponin content
The vanillin-sulphuric acid assay [27] for determining the TSC of sample materials is usually done by incubating 0.25 ml of sample extracts, standards or reagent blank with 0.25 ml of 8% (w/v) vanillin in ethanol and 2.50 ml of 72% (v/v) sulphuric acid in water for 15 min at 60 °C in a shaking water bath, with the standards and the reagent blank made up with the solvent used for extracting the plant samples (extraction solvent). After cooling in water at the ambient temperature for 5 min, the absorbance of the standards and extracts are measured at 560 nm using a Cary 50 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) after zeroing it with the reagent blank. The TSC of the samples is then expressed in mg of standard equivalents per gram of plant sample (mg SE g−1).

RESULTS
In the present study, a total of 13 endolichenic fungi were isolated from the lichen species Hypotrachyna infirma (Kurok.)Hale. Phytochemical studies including qualitative (Tannins, Flavanoids, Saponins, Steriods, Carbohydrates, Glycosides, Alkaloids, Proteins and Phenols) and quantitative (Tannins, Flavonoids, Saponins and Phenols) were done in two solvents ethyl acetate and chloroform for the isolated endolichenic fungi. From these two solvents, ethyl acetate gave best result compared to chloroform. Based on the results obtained from the qualitative analysis, the quantitative analysis was carried out. Table 1 shows qualitative analysis of 13 endolichenic fungal extracts in two solvents (ethyl acetate and Chloroform). The results of the phytochemical analysis of ethyl acetate extracts of Geotrichum candidum Link, Scytalidium lignicola Pesante, Aspergillus niger Gr, Aspergillus oryzae(Ahlb.)cohn, Nigrospora oryzae(Berk and Broome) Petch have high content of tannin, flavonoids, saponin and phenols, less content of Terpenoids, Steriods, Carbohydrates, Glycosoids, alkaloids, proteins. But Terpenoids were absent in Aspergillus oryzae(Ahlb.) cohn, and Glycosoids was absent in Geotrichum candidum Link.

Quantitative phytochemical screening
Quantitative phytochemical analysis is performed in two solvents ethyl acetate and chloroform. Graph 1 to 13 showed the presence and the amount of certain phytochemicals in the fungal extracts.

DISCUSSION
The secondary metabolites are different in various organic extracts of all the fungal culture. Their qualitative analysis revealed their appearance whereas their quantitative analysis give almost approximate idea for their quantity present Endolichenic fungi were discovered when attempts were being made to isolate the lichen forming mycobiont into pure culture [28,29]. These fungi are similar to the endophytic fungi (sometimes also referred to as endophyte-like fungi) [30,31], which reside within healthy tissues of plants and are phylogenetically and ecologically diverse without causing any disease symptoms [32,33].
The overlap between endolichenic and endophytic metabolites is consistent with their biological similarities; there exists considerable overlap in the taxa represented in endolichenic and endophytic fungal strains, and they are believed to perform similar ecological roles for the host organism [34]. However, endolichenic fungal metabolites remain relatively distinct from the natural products produced by lichens individually [35,36].
In the living systems, alkaloids are most significant compounds that play a metabolic role and are involved in the protective function in animals. Nigrospora oryzae (Berkand Broome) Petch and Geotrichum candidum (Berkand Broome) Petch gave good results. The results showed the high amount of Flavonoids and phenols present in Nigrospora oryzae(Berkand Broome) Petch>Geotrichum candidum Link>Scytalidium lignicola pesante>Aspergillus oryzae (Ahlb.) cohn>Aspergillus niger Gr. Flavonoids inhibits the promotion of growth and progression of tumors and also used against the cancercausing tumors [37]. In plants phenols when mixed with the flavonoid compounds it show multiple activities like antioxidant, anticarcinogenic, anti-inflammatory, etc. [38]. Singh and Bhat (2003) [39] studied flavonoids are responsible for the antimicrobial activity associated with some ethno medicinal plants.
The presence of tannins in extracts inhibits the pathogenic fungi and showed better antimicrobial activity [40]. The presence of tannins in diets for livestock have been showed to have antinutritional and toxic effects including fed intake, growth, feed efficiency and net metabolizable energy [41]. Nigrospora oryzae (Berk and Broome) Petch contain high amount of tannin compared to the other endolichenic fungus.
The natural compounds have an effective dosage response and minimum side effects when compared to synthetic compounds. The plant screened for phytochemical constituents seemed to have the potential function as a source of beneficial drugs and also to improve the health status of the consumers as a result of the presence of various compounds that are vital for good health [42].

CONCLUSION
From this study Nigrospora oryzae (Berk and Broome) Petch gave the best results in both qualitative and quantitative compared to other endolichenic fungi. Geotrichum candidum (Berkand Broome) Petch, Scytalidium lignicola Pesante gave the moderate results. Aspergillus niger Gr, Aspergillus oryzae (Ahlb.)cohn gave less results compared to other organisms. Based on the phytochemical studies, further research can be carried out to isolate the particular phytochemical compounds in the endolichenic fungi.